Cells contain a number of proteins known as molecular chaperones or foldases. These molecules catalyse the folding of newly synthesized proteins, prevent aggregation and improper glycosylation, and remove denatured proteins. Although they do not become part of the final structure, they are important in the assembly of proteins or their subunits into larger, more complex structures. In the absence of chaperones and foldases, misfolded proteins are quickly degraded by intracellular proteases.
The molecular chaperones include the heat shock proteins (particularly Hsp70) such as DnaK and binding protein (BiP). Both DnaK and BiP may be located in the cytoplasm where they bind to newly formed proteins as they are released from the ribosomal machinery. These chaperones prevent aggregation by binding to the "sticky" or hydrophobic surfaces of the protein molecule. The catalytic protein disulfide isomerase (PDI; also known as glycosylation site binding protein, GSBP) is a foldase (or shufflease; Laboissiere M C et al. (1995) J Biol Chem 270:28006-9) which is found in membrane-bound eukaryotic compartments such as the endoplasmic reticulum (ER). It facilitates disulfide bond exchange as well as correct glycosylation. Molecular chaperones and foldases disassociate from their protein substrates as soon as the protein has assumed its native conformation.
In prokaryotes such as E. coli, DnaK, an Hsp70 molecule, binds to partially folded cytoplasmic proteins and facilitates their folding. In E. coli, export of a partially folded protein may also be facilitated by molecular chaperone. Because protein folding is both a stoichometric and an energy requiring process, overexpression of recombinant proteins in prokaryotes commonly leads to aggregation of the protein and results in the formation of inclusion bodies.
Although a bacterial form of hsp70 is found in the mitochondria, BiP is a specialized eukaryotic Hsp70 which carries out its activities in the ER. BiP binds to hydrophobic portions of a nascent protein before the protein leaves the ribosome and hydrolyzes ATP to provide energy for the folding that allows the protein to attain its native conformation. Although the exact energy cost for protein folding is unknown, estimates range from 30-100 molecules per turnover event.
Foldases, such as PDI, are specialized enzymes which carry out rate-limiting covalent steps in protein folding. These enzymes are most abundant in cells actively synthesizing secreted proteins which are major components of the ER lumen (Tasanen K et al. (1992) J Biol Chem 267:11513-19) and may constitute 1-2% of eukaryotic cellular proteins. Although incubation of reduced unfolded proteins in buffers with defined ratios of oxidized and reduced thiols can lead to native conformation, the rate of folding is slow and the attainment of native conformation decreases proportionately to the size and number of cysteines in the protein. In contrast, PDI in the eukaryotic ER is much more efficient in carrying out the enzymatic pairing and oxidation of cysteines.
In general, disulfides are formed only in secretory compartments such as the ER or periplasmic space because the redox potential of the cytoplasm is unfavorable. The correct folding of proteins which contain disulfide bonds is also most likely to occur when the protein is expressed with an intact leader sequence which allows Its export into appropriate compartments for enzymatic processing by PDI.
LaMantia et al. (1994; Proc Nati Acad Sci 88:4453-57) first reported that PDI and GSBP were identical in yeast. Disruption of the gene in yeast experimentally resulted in a recessive lethal mutation demonstrating that PDI/GSBP activity is necessary for cell viability. Other molecules found in cells actively secreting proteins and closely related to PDI are the .beta. subunit of the tetrameric prolyl 4-hydroxylass (Pihlajaniemi T et al. (1987) EMBO J 6:643-49), a component of the triglyceride transfer protein, and a thyroid hormone binding protein (cf. Hayano T and M Kikuchi (1995) FEBS Lett 372:210-214).